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Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations 5th edition [Kõva köide]

(Pharmacia Biotech Europe GmbH, Freiburg, Germany)
  • Formaat: Hardback, 458 pages, kõrgus x laius x paksus: 249x175x28 mm, kaal: 1134 g
  • Ilmumisaeg: 07-Apr-2016
  • Kirjastus: Blackwell Verlag GmbH
  • ISBN-10: 3527338802
  • ISBN-13: 9783527338801
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Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations 5th editionSuurem pilt
  • Formaat: Hardback, 458 pages, kõrgus x laius x paksus: 249x175x28 mm, kaal: 1134 g
  • Ilmumisaeg: 07-Apr-2016
  • Kirjastus: Blackwell Verlag GmbH
  • ISBN-10: 3527338802
  • ISBN-13: 9783527338801
Teised raamatud teemal:
Püsilink: https://www.kriso.ee/db/9783527338801.html
Märksõnad:
This fifth edition of the successful, long-selling classic has been completely revised and expanded, omitting some topics on obsolete DNA electrophoresis, but now with a completely new section on electrophoretic micro-methods and on-the-chip electrophoresis.
The text is geared towards advanced students and professionals and contains extended background sections, protocols and a trouble-shooting section. It is now also backed by a supplementary website providing all the figures for teaching purposes, as well as a selection of animated figures tested in many workshops to explain the underlying principles of the different electrophoretic methods.
Foreword xix
Abbreviations, Symbols, Units xxi
Preface xxv
Part I Fundamentals 1(186)
Introduction
1(1)
Principle
1(2)
Areas of Applications
3(1)
The Sample
3(1)
The Buffer
4(1)
Electroendosmosis
5(1)
References
6(1)
1 Electrophoresis
7(50)
1.1 General
7(18)
1.1.1 Electrophoresis in Free Solution
7(5)
1.1.2 Electrophoresis in Supporting Media
12(1)
1.1.3 Gel Electrophoresis
13(6)
1.1.3.1 Gel Types
13(4)
1.1.3.2 Instrumentation for Gel Electrophoresis
17(1)
1.1.3.3 Current and Voltage Conditions
17(2)
1.1.4 Power Supply
19(1)
1.1.5 Separation Chambers
20(5)
1.1.5.1 Vertical Systems
20(1)
1.1.5.2 Horizontal Systems
21(4)
1.2 Electrophoresis in Nonrestrictive Gels
25(3)
1.2.1 Agarose Gel Electrophoresis
25(3)
1.2.1.1 Zone Electrophoresis
25(1)
1.2.1.2 Immunoelectrophoresis
26(1)
1.2.1.3 Affinity Electrophoresis
27(1)
1.2.2 Polyacrylamide Gel Electrophoresis of Low Molecular Weight Substances
28(1)
1.3 Electrophoresis in Restrictive Gels
28(23)
1.3.1 The Ferguson Plot
28(1)
1.3.2 Agarose Gel Electrophoresis
29(1)
1.3.2.1 Proteins
29(1)
1.3.2.2 Nucleic Acids
29(1)
1.3.3 Pulsed-Field Gel Electrophoresis
30(2)
1.3.4 Polyacrylamide Gel Electrophoresis of Nucleic Acids
32(5)
1.3.4.1 DNA Sequencing
32(2)
1.3.4.2 DNA Typing
34(1)
1.3.4.3 Mutation Detection Methods
35(2)
1.3.4.4 Denaturing PAGE of Microsatellites
37(1)
1.3.4.5 Two-dimensional DNA Electrophoresis
37(1)
1.3.5 Polyacrylamide Gel Electrophoresis of Proteins
37(28)
1.3.5.1 Disc Electrophoresis
37(2)
1.3.5.2 Gradient Gel Electrophoresis
39(1)
1.3.5.3 SDS Electrophoresis
40(7)
1.3.5.4 Cationic Detergent Electrophoresis
47(1)
1.3.5.5 Blue Native Electrophoresis
47(1)
1.3.5.6 Rehydrated Polyacrylamide Gels
48(1)
1.3.5.7 Two-Dimensional Electrophoresis Techniques
49(1)
1.3.5.8 GeLC-MS
50(1)
References
51(6)
2 Isotachophoresis
57(6)
2.1 Migration with the Same Speed
57(2)
2.2 "Ion Train" Separation
59(1)
2.3 Zone Sharpening Effect
59(1)
2.4 Concentration Regulation Effect
59(1)
2.5 Quantitative Analysis
60(1)
References
61(2)
3 Isoelectric Focusing
63(22)
3.1 Principles
63(2)
3.2 Gels for IEF
65(3)
3.2.1 Polyacrylamide Gels
65(2)
3.2.2 Agarose Gels
67(1)
3.3 Temperature
68(1)
3.4 Controlling the pH Gradient
68(1)
3.5 Kinds of pH Gradients
69(8)
3.5.1 Free Carrier Ampholytes
69(4)
3.5.1.1 Electrode Solutions
70(1)
3.5.1.2 Denaturing IEF: Urea IEF
71(1)
3.5.1.3 Separator IEF
72(1)
3.5.1.4 Plateau Phenomenon
73(1)
3.5.1.5 The Workflow of a Carrier Ampholyte IEF Run
73(1)
3.5.2 Immobilized pH Gradients (IPG)
73(4)
3.5.2.1 Preparation of Immobilized pH Gradients
75(1)
3.5.2.2 Applications of Immobilized pH Gradients
76(1)
3.6 Protein Detection in IEF Gels
77(1)
3.7 Preparative Isoelectric Focusing
77(3)
3.7.1 Carrier Ampholyte IEF in Gel
77(1)
3.7.2 Carrier Ampholyte IEF in Free Solution
78(1)
3.7.3 Immobilized pH Gradients
78(7)
3.7.3.1 Isoelectric Membranes
78(1)
3.7.3.2 Off-Gel IEF
79(1)
3.8 Titration Curve Analysis
80(2)
References
82(3)
4 High-Resolution Two-Dimensional Electrophoresis
85(26)
4.1 IEF in Immobilized pH Gradient Strips
85(13)
4.1.1 Strip Lengths
86(1)
4.1.2 pH Gradient Types
86(1)
4.1.3 The Influence of Salts and Buffer Ions on the Separation
87(1)
4.1.4 Basic IPG Gradients
88(1)
4.1.5 Advantages of Immobilized pH Gradient Strips in 2D Electrophoresis
89(1)
4.1.6 Rehydration of IPG Strips
90(2)
4.1.6.1 Basic pH Gradients
90(1)
4.1.6.2 Reswelling Tray
91(1)
4.1.6.3 Cover Fluid
91(1)
4.1.6.4 Rehydration Time
92(1)
4.1.7 Sample Application on IPG Strips
92(3)
4.1.8 IEF Conditions
95(1)
4.1.8.1 Electrode Pads
95(1)
4.1.8.2 Temperature
95(1)
4.1.8.3 Electric Conditions
95(1)
4.1.8.4 Time
96(1)
4.1.9 Instrumentation
96(2)
4.1.9.1 The Strip Tray Accessory
97(1)
4.1.9.2 Dedicated Instruments for IPG Strips
97(1)
4.1.9.3 Running IEF in IPG Strips
97(1)
4.2 SDS-PAGE
98(8)
4.2.1 Equilibration of the IPG Strips
98(1)
4.2.2 Technical Concepts for the Second Dimension (SDS-PAGE)
99(2)
4.2.2.1 Vertical Set-ups
99(1)
4.2.2.2 Horizontal Set-ups
99(2)
4.2.3 Gel Types
101(1)
4.2.3.1 Gel Sizes
101(1)
4.2.3.2 Vertical Gels
101(1)
4.2.3.3 Horizontal Gels
102(1)
4.2.4 Gel Casting
102(3)
4.2.4.1 Gels for Multiple Vertical Systems
102(2)
4.2.4.2 Gels for Horizontal Systems
104(1)
4.2.5 Running the SDS Gels
105(8)
4.2.5.1 Vertical Systems
105(1)
4.2.5.2 Horizontal Systems
106(1)
4.3 Proteomics
106(2)
References
108(3)
5 Protein Sample Preparation
111(20)
5.1 Protein Quantification Methods
111(1)
5.2 Preparation of Native Samples
112(1)
5.3 Samples for SDS Electrophoresis
113(5)
5.3.1 SDS Treatment
113(4)
5.3.1.1 Nonreducing SDS Treatment
114(1)
5.3.1.2 Reducing SDS Treatment
115(1)
5.3.1.3 Reducing SDS Treatment with Subsequent Alkylation
116(1)
5.3.2 Clean-up and Protein Enrichment
117(1)
5.3.2.1 Precipitation
117(1)
5.3.2.2 Protein Enrichment by Affinity Beads
118(1)
5.4 Samples for High-Resolution 2D PAGE
118(9)
5.4.1 Cell Washing
119(1)
5.4.2 Cell Disruption
119(1)
5.4.3 Sample Acquisition and Storage
119(3)
5.4.4 Protease Inactivation
122(1)
5.4.5 Phosphatase Inactivation
122(1)
5.4.6 Alkaline Conditions
123(1)
5.4.7 Removal of Contaminants
123(2)
5.4.7.1 Precipitation Methods
123(2)
5.4.7.2 Affinity Beads
125(1)
5.4.8 Prefractionation
125(2)
5.4.8.1 Depletion of Highly Abundant Proteins
125(1)
5.4.8.2 Equalizer Technology
125(1)
5.4.8.3 Preseparation of Cell Organelles
126(1)
5.4.8.4 Prefractionation according to Isoelectric Points
126(1)
5.4.9 Special Case: Plant Proteins
127(1)
References
127(4)
6 Protein Detection
131(34)
6.1 Fixation
131(2)
6.1.1 IEF Gels
132(1)
6.1.2 Agarose Gels
132(1)
6.1.3 SDS Polyacrylamide Gels
132(1)
6.2 Poststaining Methods
133(7)
6.2.1 Organic Dyes
133(1)
6.2.1.1 Monodisperse Coomassie Brilliant Blue Staining
133(1)
6.2.1.2 Colloidal Coomassie Brilliant Blue Staining
133(1)
6.2.1.3 Acid Violet 17 Staining for IEF Gels
134(1)
6.2.2 Silver Staining
134(2)
6.2.2.1 Colloidal Silver Staining
134(1)
6.2.2.2 Silver Nitrate Staining
134(1)
6.2.2.3 Ammoniacal Silver Staining
135(1)
6.2.3 Negative Staining
136(1)
6.2.3.1 Copper Staining
136(1)
6.2.3.2 Imidazole Zinc Staining
136(1)
6.2.4 Fluorescent Staining
136(2)
6.2.5 Specific Detection
138(2)
6.2.5.1 Proteins with Posttranslational Modifications
138(1)
6.2.5.2 Isoenzymes
139(1)
6.2.6 Stain-Free Technology
140(1)
6.3 Prelabeling
140(3)
6.3.1 Prelabeling with Fluorescent Tags
140(1)
6.3.2 Radioactive Labeling of Living Cells
141(1)
6.3.3 Labeling with Stable Isotopes
141(2)
6.4 Difference Gel Electrophoresis (DIGE)
143(6)
6.4.1 Minimum Lysine Labeling
143(1)
6.4.2 Saturation Cysteine Labeling
144(2)
6.4.3 The Internal Standard
146(1)
6.4.4 Experimental Design
147(1)
6.4.5 Major Benefits of 2D DIGE
147(1)
6.4.6 Specific Labeling of Cell-Surface Proteins
148(1)
6.4.7 Comparative Fluorescence Gel Electrophoresis
148(1)
6.5 Imaging, Image Analysis, Spot Picking
149(11)
6.5.1 Quantitative Evaluation
149(2)
6.5.1.1 Quantification Prerequisites
149(1)
6.5.1.2 Critical Issues in Quantification
150(1)
6.5.2 Imaging Systems
151(3)
6.5.2.1 Optical Density
152(1)
6.5.2.2 Densitometry
152(1)
6.5.2.3 CCD Cameras
153(1)
6.5.3 Image Analysis
154(4)
6.5.3.1 One-Dimensional Gel Software
155(1)
6.5.3.2 Two-Dimensional Gel Software
156(2)
6.5.4 Protein Identification and Characterization
158(7)
6.5.4.1 Spot-Picking
159(1)
References
160(5)
7 Blotting
165(22)
7.1 Transfer Methods
165(6)
7.1.1 Diffusion Blotting
165(1)
7.1.2 Capillary Blotting
165(1)
7.1.3 Pressure Blotting
166(1)
7.1.4 Vacuum Blotting
167(1)
7.1.5 Electrophoretic Blotting
168(4)
7.1.5.1 Tank Blotting
168(1)
7.1.5.2 Semidry Blotting
169(2)
7.1.5.3 Electrophoretic Blotting of Film-Backed Gels
171(1)
7.2 Blotting Membranes
171(1)
7.3 Buffers for Electrophoretic Transfers
172(2)
7.3.1 Proteins
172(2)
7.3.1.1 Tank Blotting
172(1)
7.3.1.2 Semidry Blotting
173(1)
7.3.2 Nucleic Acids
174(1)
7.3.2.1 Tank Blotting
174(1)
7.3.2.2 Semidry Blotting
174(1)
7.4 General Staining
174(1)
7.5 Blocking
175(1)
7.6 Specific Detection
175(5)
7.6.1 Hybridization
175(1)
7.6.2 Enzyme Blotting
176(1)
7.6.3 Immunoblotting
176(3)
7.6.4 Lectin Blotting
179(1)
7.6.5 Stripping, Reprobing
179(1)
7.6.6 Double Blotting
180(1)
7.7 Protein Sequencing
180(1)
7.8 Transfer Issues
180(1)
7.9 Electro-Elution of Proteins from Gels
181(2)
References
183(4)
Part II Equipment and Methods 187(206)
Equipment
187(1)
Methods
187(1)
Small Molecules
187(1)
Proteins
187(1)
DNA
188(1)
Instrumentation
188(1)
Accessories
189(1)
Consumables
190(1)
8 Special Laboratory Equipment
191(2)
9 Consumables
193(2)
10 Chemicals
195(2)
10.1 Reagents
195(2)
Method 1 PAGE of Dyes
197(8)
M1.1 Sample Preparation
197(1)
M1.2 Stock Solutions
197(1)
M1.3 Preparing the Casting Cassette
198(2)
M1.3.1 Gasket
198(1)
M1.3.2 Slot-Former
198(1)
M1.3.3 Assembling the Gel Cassette
199(1)
M1.4 Casting Ultra-Thin-Layer Gels
200(1)
M1.5 Electrophoretic Separation
201(4)
M1.5.1 Removing the Gel from the Cassette
201(4)
Method 2 Agarose and Immunoelectrophoresis
205(12)
M2.1 Sample Preparation
205(1)
M2.2 Stock Solutions
206(1)
M2.3 Preparing the Gels
206(5)
M2.3.1 Agarose Gel Electrophoresis
206(3)
M2.3.1.1 Preparing the Slot-Former
207(1)
M2.3.1.2 Assembling the Gel Cassette
207(2)
M2.3.2 Immunoelectrophoresis Gels
209(2)
M2.3.2.1 Punching Out the Sample Wells and Troughs
210(1)
M2.4 Electrophoresis
211(3)
M2.4.1 Grabar -Williams Technique
212(1)
M2.4.2 Laurell Technique
212(2)
M2.5 Protein Detection
214(2)
M2.5.1 Coomassie Staining (Agarose Electrophoresis)
214(1)
M2.5.2 Immunofixing of Agarose Electrophoresis
214(1)
M2.5.3 Coomassie Staining (Immunoelectrophoresis)
215(1)
M2.5.4 Silver Staining
215(1)
References
216(1)
Method 3 Titration Curve Analysis
217(12)
M3.1 Sample Preparation
217(1)
M3.2 Stock Solutions
217(1)
M3.3 Preparing the Blank Gels
218(4)
M3.3.1 Preparing the Casting Cassette
218(1)
M3.3.2 Assembling the Gel Cassette
219(1)
M3.3.3 Filling the Gel Cassette
220(1)
M3.3.4 Removing the Gel from the Cassette
221(1)
M3.3.5 Washing the Gel
221(1)
M3.4 Titration Curve Analysis
222(2)
M3.4.1 Reswelling the Rehydratable Gel
222(1)
M3.4.2 Formation of the pH Gradient
222(1)
M3.4.3 Native Electrophoresis in the pH Spectrum
223(1)
M3.5 Coomassie and Silver Staining
224(1)
M3.5.1 Colloidal Coomassie Staining
224(1)
M3.5.2 Acid Violet 17 Staining
224(1)
M3.5.3 Five-Minute Silver Staining of Dried Gels
225(1)
M3.6 Interpreting the Curves
225(2)
References
227(2)
Method 4 Native PAGE in Amphoteric-Buffers
229(14)
M4.1 Sample Preparation
230(1)
M4.2 Stock Solutions
230(1)
M4.3 Preparing the Empty Gels
231(4)
M4.3.1 Slot-Former
231(1)
M4.3.2 Assembling the Casting Cassette
232(1)
M4.3.3 Polymerization Solutions
233(1)
M4.3.4 Filling the Cooled Gel Cassette
234(1)
M4.3.5 Removing the Gel from the Casting Cassette
234(1)
M4.3.6 Washing the Gel
234(1)
M4.4 Electrophoresis
235(5)
M4.4.1 Rehydration in Amphoteric Buffers
235(5)
M4.5 Coomassie and Silver Staining
240(2)
M4.5.1 Colloidal Coomassie Staining
240(1)
M4.5.2 Acid Violet 17 Staining
240(1)
M4.5.3 Five-Minute Silver Staining of Dried Gels
241(1)
References
242(1)
Method 5 Agarose IEF
243(10)
M5.1 Sample Preparation
243(1)
M5.2 Preparing the Agarose Gel
244(3)
M5.2.1 Making the Spacer Plate Hydrophobic
244(1)
M5.2.2 Assembling the Casting Cassette
244(2)
M5.2.3 Preparation of Electrode Solutions
246(1)
M5.3 Isoelectric Focusing
247(2)
M5.4 Protein Detection
249(2)
M5.4.1 Coomassie Blue Staining
249(1)
M5.4.2 Immunofixation
249(1)
M5.4.3 Silver Staining
250(1)
References
251(2)
Method 6 PAGIEF in Rehydrated Gels
253(14)
M6.1 Sample Preparation
253(1)
M6.2 Stock Solutions
254(1)
M6.3 Preparing the Blank Gels
254(3)
M6.3.1 Making the Spacer Plate Hydrophobic
254(1)
M6.3.2 Assembling the Casting Cassette
255(1)
M6.3.3 Filling the Gel Cassette
256(1)
M6.3.4 Removing the Gel from the Casting Cassette
257(1)
M6.3.5 Washing the Gel
257(1)
M6.4 Isoelectric Focusing
257(3)
M6.4.1 Rehydration Solution with Carrier Ampholytes (SERVALYT™, Pharmalyte™)
257(1)
M6.4.2 Reswelling the Gel
257(2)
M6.4.3 Separation of Proteins
259(1)
M6.4.4 Sample Application
259(1)
M6.5 Coomassie and Silver Staining
260(4)
M6.5.1 Colloidal Coomassie Staining
260(1)
M6.5.2 Acid Violet 17 Staining
261(1)
M6.5.3 Five-Minute Silver Staining of Dried Gels
261(1)
M6.5.4 The Most Sensitive Silver Staining Procedure for IEF
262(2)
M6.6 Perspectives
264(2)
References
266(1)
Method 7 Horizontal SDS-PAGE
267(22)
M7.1 Sample Preparation
267(2)
M7.1.1 Nonreducing SDS Treatment
267(1)
M7.1.2 Reducing SDS Treatment
268(1)
M7.1.3 Reducing SDS Treatment with Alkylation
268(1)
M7.2 Prelabeling with Fluorescent Dye
269(1)
M7.2.1 Labeling
269(1)
M7.2.2 Detection
269(1)
M7.3 Stock Solutions for Gel Preparation
270(1)
M7.4 Preparing the Casting Cassette
271(2)
M7.4.1 Preparing the Slot-Former
271(1)
M7.4.2 Assembling the Casting Cassette
272(1)
M7.5 Gradient Gel
273(4)
M7.5.1 Pouring the Gradient
273(4)
M7.6 Electrophoresis
277(2)
M7.6.1 Preparing the Separation Chamber
277(1)
M7.6.2 Placing the Gel on the Cooling Plate
277(1)
M7.6.3 Electrophoresis
278(1)
M7.7 Protein Detection
279(5)
M7.7.1 Hot Coomassie Staining
279(1)
M7.7.2 Colloidal Staining
280(1)
M7.7.2.1 Stock Solutions
280(1)
M7.7.2.2 Fixation Solution
280(1)
M7.7.2.3 Staining Solution
280(1)
M7.7.2.4 Staining Procedure
281(1)
M7.7.3 Reversible Imidazole- Zinc Negative Staining
281(1)
M7.7.4 Silver Staining
281(2)
M7.7.4.1 Blue Toning
282(1)
M7.7.5 Fluorescent Staining with SERVA Purple
283(2)
M7.7.5.1 Stock Solutions
283(1)
M7.7.5.2 Staining Protocol
283(1)
M7.7.5.3 Detection
284(1)
M7.8 Blotting
284(1)
M7.9 Perspectives
285(2)
M7.9.1 Gel Characteristics
285(1)
M7.9.2 SDS Electrophoresis in Washed and Rehydrated Gels
285(1)
M7.9.3 SDS Disc Electrophoresis in a Rehydrated and Selectively Equilibrated Gel
285(1)
M7.9.4 Peptide Separation
286(1)
References
287(2)
Method 8 Vertical PAGE
289(22)
M8.1 Sample Preparation and Prelabeling
290(1)
M8.2 Stock Solutions for SDS- PAGE
290(1)
M8.3 Single Gel Casting
291(4)
M8.3.1 Discontinuous SDS-Polyacrylamide Gels
292(1)
M8.3.2 Porosity Gradient Gels
293(2)
M8.4 Multiple Gel Casting
295(4)
M8.4.1 Multiple Discontinuous SDS Polyacrylamide Gels
296(2)
M8.4.2 Multiple SDS Polyacrylamide Gradient Gels
298(1)
M8.5 Electrophoresis
299(2)
M8.5.1 Running Conditions
300(1)
M8.6 SDS Electrophoresis of Small Peptides
301(2)
M8.7 Blue Native PAGE
303(3)
M8.8 Two-Dimensional Electrophoresis
306(1)
M8.9 DNA Electrophoresis
307(1)
M8.10 Long-Shelf-Life Gels
308(1)
M8.11 Protein Detection
308(1)
M8.12 Preparing Glass Plates with Bind-Silane
308(2)
M8.12.1 Coating a Glass Plate with Bind-Silane
309(1)
M8.12.2 Removal of Gel and Bind-Silane from a Glass Plate
309(1)
References
310(1)
Method 9 Semidry Blotting of Proteins
311(10)
M9.1 Transfer Buffers
313(1)
M9.2 Technical Procedure
314(4)
M9.2.1 Gels Without Support Film
315(1)
M9.2.2 Gels on Film Backing
315(8)
M9.2.2.1 Using a Nitrocellulose (NC) Blotting Membrane
316(1)
M9.2.2.2 Using a PVDF Blotting Membrane
316(1)
M9.2.2.3 Transfer from Cut-Off Gels
317(1)
M9.3 Staining of Blotting Membranes
318(2)
References
320(1)
Method 10 IEF in Immobilized pH Gradients
321(20)
M10.1 Sample Preparation
322(1)
M10.2 Stock Solutions
322(1)
M10.3 Immobiline Recipes
323(4)
M10.3.1 Custom-Made pH Gradients
323(4)
M10.4 Preparing the Casting Cassette
327(1)
M10.4.1 Making the Spacer Plate Hydrophobic
327(1)
M10.4.2 Assembling the Casting Cassette
327(1)
M10.5 Preparing the pH Gradient Gels
328(4)
M10.5.1 Pouring the Gradient
328(4)
M10.5.1.1 Setting Up the Casting Apparatus
328(1)
M10.5.1.2 Filling the Cassette
329(2)
M10.5.1.3 Washing the Gel
331(1)
M10.5.1.4 Storage
332(1)
M10.5.1.5 Rehydration
332(1)
M10.6 Isoelectric Focusing
332(4)
M10.6.1 Placing the Gel on the Cooling Plate
332(3)
M10.6.2 Sample Application
335(1)
M10.6.3 Electrode Solutions
335(1)
M10.6.4 Focusing Conditions
335(1)
M10.6.5 Measuring the pH Gradient
336(1)
M10.7 Staining
336(1)
M10.7.1 Colloidal Coomassie Staining
336(1)
M10.7.2 Acid Violet 17 Staining
337(1)
M10.7.3 Staining Procedure
337(1)
M10.7.4 Silver Staining
337(1)
M10.7.5 Practical Tip
337(1)
M10.8 Strategies for IPG Focusing
337(2)
References
339(2)
Method 11 High-Resolution 2D Electrophoresis
341(38)
M11.1 Sample Preparation
342(4)
M11.1.1 Sample Clean-Up
343(3)
M11.2 Prelabeling of Proteins with Fluorescent Dyes
346(6)
M11.2.1 Labeling of One Sample
346(1)
M11.2.2 DIGE Labeling
347(7)
M11.2.2.1 Experimental Design
347(1)
M11.2.2.2 Sample Preparation
347(1)
M11.2.2.3 Reconstitution of the CyDyes
348(1)
M11.2.2.4 Minimal Labeling of the Lysines
349(1)
M11.2.2.5 Saturation Labeling of the Cysteines
350(1)
M11.2.2.6 Preparation for Loading the Samples onto the IPG Strips
351(1)
M11.2.2.7 Detection of DIGE Spots
352(1)
M11.3 Stock Solutions for Gel Preparation
352(2)
M11.4 Preparing the Gels
354(5)
M11.4.1 IPG Strips
354(4)
M11.4.2 SDS Polyacrylamide Gels
358(1)
M11.5 Separation Conditions
359(11)
M11.5.1 First Dimension (IPG-IEF)
359(7)
M11.5.1.1 IPG-IEF with Conventional Equipment
360(1)
M11.5.1.2 IPG-IEF with IPG Strip Kit (Figure)
360(2)
M11.5.1.3 IPG-IEF in Individual Ceramic Trays
362(1)
M11.5.1.4 Equipment and Trays for Cup Loading
363(3)
M11.5.2 Equilibration
366(1)
M11.5.3 Second Dimension (SDS Electrophoresis)
366(4)
M11.5.3.1 Vertical Gels
366(1)
M11.5.3.2 Horizontal Gels
367(3)
M11.6 Staining Procedures
370(7)
M11.6.1 Staining of Multiple Gels
371(1)
M11.6.2 Colloidal Coomassie Staining
371(1)
M11.6.2.1 Stock Solutions
371(1)
M11.6.2.2 Fixation Solution
372(1)
M11.6.2.3 Staining Solution
372(1)
M11.6.2.4 Staining Procedure:
372(1)
M11.6.3 Reversible Imidazole- Zinc Negative Staining
372(1)
M11.6.4 Silver Staining
373(1)
M11.6.4.1 Mass Spectrometry Analysis of Silver-Stained Spots
374(1)
M11.6.4.2 Blue Toning
374(1)
M11.6.5 Fluorescent Staining with SERVA Purple
374(21)
M11.6.5.1 Stock Solutions
374(1)
M11.6.5.2 Staining Protocol
375(1)
M11.6.5.3 Detection
376(1)
References
377(2)
Method 12 PAGE of DNA Fragments
379(14)
M12.1 Stock Solutions
380(1)
M12.2 Preparing the Gels
381(4)
M12.3 Sample Preparation
385(1)
M12.4 Electrophoresis
386(5)
M12.5 Silver Staining
391(2)
Appendix Troubleshooting 393(42)
A1.1 Frequent Mistakes
393(3)
A1.1.1 Miscalculation of the Cross-Linking Factor of a Polyacrylamide Gel
393(1)
A1.1.2 Polymerization Temperature and Time for a Polyacrylamide Gel
393(1)
A1.1.3 Creating Aggregates in SDS Samples
394(1)
A1.1.4 Titration of the Running Buffer in SDS Electrophoresis
394(1)
A1.1.5 Incomplete Removal of PBS from Cells
395(1)
A1.1.6 Over-focusing of IPG Strips in 2D PAGE
395(1)
A1.1.6.1 Protein Degradation in Basic pH Gradients
395(1)
A1.1.6.2 The "Thiourea Effect"
395(1)
A1.2 Isoelectric Focusing
396(14)
A1.2.1 PAGIEF with Carrier Ampholytes
396(6)
A1.2.2 Agarose IEF with Carrier Ampholytes
402(3)
A1.2.3 Immobilized pH Gradients
405(5)
A1.3 SDS Electrophoresis
410(9)
A1.3.1 Horizontal SDS-PAGE
410(8)
A1.3.2 Vertical PAGE
418(1)
A1.4 Two-Dimensional Electrophoresis
419(7)
A1.5 Semi-Dry Blotting
426(5)
A1.6 DNA Electrophoresis
431(4)
Index 435
Reiner Westermeier worked after PhD graduation and Post-doc at the Technische Universitat Munchen for 30 years as a specialist for electrophoresis methods for leading bioanalytics and biotechnology companies. His area of responsibility included co-operation in product development, writing of scientic papers and method instructions, trouble shooting in customer laboratories, performing seminars and practical courses on electrophoresis and proteomics, as well as giving scientific talks at congresses on a world-wide basis. He is editor and author of several books, e.g. 'Electrophoresis in Practice' (in German and in English), 'Proteomics in Practice', and 'Difference Gel Electrophoresis'.